Abstract
BACKGROUND: The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production. RESULTS: This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing P(gpdA)) mediated by the SP(CAT), a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by P(citA)-SP(GlaA) had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing P(citA)-SP(GlaA)) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu(2+) in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter. CONCLUSIONS: To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SP(CAT) in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger.